1

α1-acid glycoprotein attenuates adriamycin-induced nephropathy via CD163

expressing macrophages induction

Rui Fujimura1,2,*, Hiroshi Watanabe1,*,#, Kento Nishida1, Yukio Fujiwara3, Tomoaki

Koga4, Jing Bi1,2, Tadashi Imafuku1,2, Kazuki Kobayashi1, Hisakazu Komori1, Masako

Miyahisa1, Hitoshi Maeda1, Motoko Tanaka5, Kazutaka Matsushita5, Takashi Wada6,

Masafumi Fukagawa7, Toru Maruyama1,#

1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences,

Kumamoto University, 5-1 Oe-Honmachi, Chuo-ku, Kumamoto 862-0973, Japan

2Program for Leading Graduate Schools "HIGO (Health life science: Interdisciplinary

and Glocal Oriented) Program", Kumamoto University, 5-1 Oe-Honmachi, Chuo-ku,

Kumamoto 862-0973, Japan

3Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto

University, 1-1-1, Honjo, Chuo-ku, Kumamoto 860-8556, Japan.

4Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics,

Kumamoto University, Kumamoto, Japan

5Department of Nephrology, Akebono Clinic, 1-1 Shirafuji 5 Chome, Minami-ku,

Kumamoto 861-4112, Japan

6Department of Nephrology and Laboratory Medicine, Faculty of Medicine, Institute of

Medical, Pharmaceutical and Health Sciences, Graduate School of Medical Sciences,

Kanazawa University, 13-1, Takara-machi, Kanazawa 920-8641, Japan

7Division of Nephrology, Endocrinology and Metabolism, Tokai University School of

Kidney360 Publish Ahead of Print, published on March 24, 2020 as doi:10.34067/KID.0000782019

Copyright 2020 by American Society of Nephrology.

2

Medicine, Isehara, Kanagawa 259-1193, Japan

*equal contribution: RF and HW

#Corresponding authors:

Hiroshi Watanabe, Ph.D.; Department of Biopharmaceutics, Graduate School of

Pharmaceutical Sciences, Kumamoto University, 5-1, Oe-honmachi, Chuo-ku,

Kumamoto 862-0973, Japan; E-mail: hnabe@kumamoto-u.ac.jp

Toru Maruyama, Ph.D.; Department of Biopharmaceutics, Graduate School of

Pharmaceutical Sciences, Kumamoto University, 5-1, Oe-honmachi, Chuo-ku,

Kumamoto 862-0973, Japan; E-mail: tomaru@gpo.kumamoto-u.ac.jp

3

Abstract

Background: Recent clinical studies have shown that proteinuria is a critical factor in

the progression of chronic kidney disease (CKD) and onset of cardiovascular disease

(CVD). Inflammation and infiltration of macrophages into renal tissue are implicated as

a cause of proteinuria. α1-acid glycoprotein (AGP), an acute phase plasma protein, is

leaked into the urine in the patients with proteinuria. However, the relationship among

the urinary leakage of AGP, renal inflammation and proteinuria remains unclear.

Methods: Human AGP (hAGP) was exogenously administrated for five consecutive days

to adriamycin-induced nephropathy model mice.

Results: Adriamycin treatment increased urinary AGP accompanied by decreasing

plasma AGP in mice. Exogenous hAGP administration to adriamycin-treated mice

suppressed proteinuria, renal histological injury and inflammation. Then, hAGP

administration increased renal CD163 expression, a marker of anti-inflammatory

macrophages. Similar changes was observed in phorbol 12-myristate 13-acetate-

differentiated THP-1 cells treated with hAGP. Even in the presence of lipopolysaccharide,

hAGP treatment increased CD163/IL-10 expression in differentiated THP-1 cells.

Conclusions: AGP alleviates proteinuria and renal injury in mice with proteinuric kidney

disease via induction of CD163-expressing macrophages with anti-inflammatory function.

The results demonstrate that endogenous AGP could work to protect against glomerulus

disease. Thus, AGP supplementation could be a possible new therapeutic intervention for

patients with glomerular disease.

4

Key words: proteinuria, macrophage polarization, chronic kidney disease, alpha-1 acid

glycoprotein

5

Introduction

Approximately 13% of the worldwide population suffer from chronic kidney disease

(CKD), and the number of patients is increasing year by year.1 CKD not only leads to

end-stage renal disease (ESRD), but is also associated with a high risk of death from

cardiovascular disease (CVD).2 In previous clinical trials, proteinuria has been reported

as a critical factor involved in the progression of CKD and onset of CVD.2-4 In fact,

nephrotic patients with massive proteinuria have a 5.5-fold higher risk of CVD than

matched controls.5 It has been reported that inflammation and infiltration of immune cells

into renal tissue is a cause of proteinuria. In addition, the onset of proteinuria further

induces inflammation in tubular cells.6, 7 Therefore, it is important to stop the vicious

cycle induced by persistent proteinuria and inflammation for the prevention of CKD

progression and onset of CVD.

Recently, the involvement of macrophages in inflammation in renal pathologies has

been reported.8, 9 Macrophages have phenotypes that are classed as M1 macrophages

(classically activated macrophage) and M2 macrophages (alternatively activated

macrophage). In general, M1 macrophages promote inflammation and play a central role

in host defense during infection. In contrast, M2 macrophages are involved in anti-

inflammation and tissue remodeling.10 These phenotypes can be changed in response to

disease state. M1 macrophages predominate in the early injury phase, while M2

macrophages predominate in the recovery and repair phase.11 Indeed, previous reports

demonstrated that administration of M2 macrophages induced by IL-4 or IL-4/IL-13 was

effective against renal injury resulting from ischemia reperfusion or adriamycin-induced

6

nephropathy in mice.11, 12 These studies suggest that regulation of macrophage phenotype

in kidney could be important in reducing renal inflammation and proteinuria.

α1-acid glycoprotein (AGP), also known as orosomucoid, is a heavily glycosylated

serum protein with five branched N-glycans containing negatively charged sialic acids.13,

14 Therefore, AGP has the lowest pI value of all plasma proteins (pI =2.7-3.2). In addition,

AGP is classified as one of the major acute phase proteins in mammals and is known that

its serum concentration increases 2 to 5 fold during inflammation.15 In the patients with

proteinuria, it is observed that AGP leakes into urine.16, 17 On the other hands, AGP has

reported to possesses unique biological activities. For example, in vitro experiments have

demonstrated that AGP modulates the activation of neutrophils, lymphocytes and

monocytes.14, 18-21 Daemen et al. reported that exogenously administered AGP attenuates

renal ischemia reperfusion injury in vivo,22 even though its renoprotective mechanisms

remain unclear. Importantly, we have previously found that AGP weakly activates the

TLR4/CD14 pathway and increases the expression of CD163, a cysteine-rich scavenger

receptor in macrophages, in particular of the M2 phenotype.23, 24 Interestingly, CD163-

overexpressing macrophages inhibit LPS-induced inflammation in vitro.25 Furuhashi et

al. also found that CD163-expressing macrophages are associated with IL-10 production

as an anti-inflammatory cytokine in anti-GBM glomerulonephritis rat.26 These findings

lead to the idea that AGP potentiates to suppress the renal inflammation and proteinuria

by changing the macrophage phenotype to one with enhanced CD163 expression.

The purpose of this study is to clarify whether exogenously administered human AGP

(hAGP) attenuates renal damage in adriamycin-induced nephropathy mice. In addition,

7

possible mechanisms of this renoprotective effect are examined. Here, we provide

evidence that administration of hAGP induces CD163-expressing macrophages in kidney,

which leads to reducing proteinuria and renal inflammation in adriamycin-induced

nephropathy mice.

8

Materials and Methods

Purification of AGP

hAGP was purified from supernatant of human plasma fraction V donated by KM

Biologics (Kumamoto, Japan). Supernatant was diluted with acetate buffer (10 mM) and

injected onto an ion exchange column（GE healthcare, Little Chalfont, UK, Hitrap

CM,Q column）at a flow rate of 3 mL/min on an AKTAprime Plus System (GE

healthcare). The bound materials were eluted with acetate buffer (10 mM) containing

NaCl (0.5 mol/L). hAGP was eluted in the first peak. The eluted sample was dialyzed

against distilled water at 4 ºC, freeze-dried and stored at -20 ºC.27 The purity of hAGP

was confirmed by SDS-PAGE. The secondary structure of hAGP was confirmed by

Circular dichroism.

Animal studies

All animal experiments were performed according to the guidelines, principles, and

procedures for the care and use of laboratory animals of Kumamoto University. All

animals were housed in an environment with controlled temperature, a 12 hr light/dark

cycle and free access to food and water.

Six-week-old male BALB/c mice were obtained from Japan SLC, Inc (Shizuoka, Japan).

After randomizing mice by body weight, mice were slowly injected intravenously with

saline or adriamycin (ADR) (15 mg/kg body weight). The first administration of saline

or AGP (5 mg/mice) was intraperitoneally administered between 30 min and 1 hr after

ADR injection, allowing for time for ADR to disappear from the blood. Subsequently,

saline or hAGP were administered at the same time for 4 consecutive days. No mice were

9

died in this experimental condition. On day 7 and day 21 after ADR administration, urine

was collected by metabolic cage for a total of 16 hr from 16:00 to 8:00, and urine volume

was measured. Mice were sacrificed at day 7 or day 21 after ADR administration. Kidney

and liver were harvested after perfusion with 0.9% NaCl. A part of the left kidney was

used for quantitative RT-PCR and histological analysis.

Renal function

Urinary albumin was measured by ELISA (Shibayagi, Japan). BUN, creatinine and total

cholesterol were measured using FUJI DRI-CHEM (FUJIFILM, Japan).

Histological analysis

Sections of renal tissue were stained with Periodic acid-Schiff (PAS). Quantitative

evaluation of glomerulosclerosis and damaged tubules was performed according to the

report of Qi Cao et al.28 Images and analysis were performed using a Keyence BZ-X710

microscope. (Keyence, Osaka, Japan) The images were randomly acquired, with 12 to 17

high power fields collected for each mouse and then 21 glomeruli randomly traced within

them. The percentage of glomerulosclerosis was computed from the area which was PAS-

positive in the total area of the same glomerulus. For analysis of damaged tubules, 10

high power field images were randomly acquired of the renal cortex of each mouse. Three

people counted damaged tubules which revealed intraluminal casts, vacuolization and

droplets, and the percentage of damaged tubules was calculated using the number of

damaged tubules divided by the total number of tubules. This analysis was performed in

10

a blind manner.

Immunostaining assay

Infiltration of macrophages was confirmed by immunostaining used anti-EMR1 antibody

(F4/80, DAKO, Santa Clara, CA). 10 to 12 high power fields were randomly imaged for

each mouse, and the number of F4/80+ cells counted. The expression of nephrin and the

renal localization of AGP were confirmed by immunofluorescence using nephrin and

ORM1 antibody. Deparaffinized sections were inactivated with Histo VT One (Nacalai

Tesque, Kyoto, Japan) and incubated with nephrin antibody（1:100, R&D systems, MN）

or ORM1 antibody (1:50, Proteintech, IL) overnight at 4 ºC, followed by incubation with

Alexa 488 donkey anti-goat IgG(H+L) (1:500, Abcam plc, Cambridge, UK) or Alexa 546

goat anti-rabbit IgG(H+L) (1:200, Thermo Scientific, Waltham, MA) for 90 min at room

temperature. Images were obtained on a Keyence BZ-X710 microscope (Keyence).

Kidney cell suspension

Kidney cell suspension for isolating renal macrophages was prepared according to the

report of Qi Cao et al29. Kidney were harvested after perfusion with HBSS (without

calcium) and DMEM containing 1mg/mL collagenase IV and 100 µg/mL DNase I

(Worthington Biochemical Corp, Lakewood, NJ). Kidney tissues were cut into paste and

shaking for 40 min at 37 ºC in DMEM containing collagenase IV and DNase I. Cell

suspensions were passed through a 75 µm stainless mesh and centrifuged at 180 g for 3

min. The pellets were suspended in RBC lysis and centrifuged at 300 g for 5 min. After

11

wash using PBS(-), cell suspensions were incubated for 15 min at 4 ºC in blocking buffer

(2% FBS, 2mM EDTA) containing 10 µg/mL Fc blocker. Cell suspensions were stained

with anti-CD45.2, F4/80, and antibodies to T cell, B cell, and natural killer (NK) cell

lineages (TCRβ, TCRγδ, CD3ε, CD19, CD49b).

Isolation of renal macrophages

Kidney cell suspensions were gated on FSC and SSC to remove doublet, and exclude

dead cells using propidium iodide. Then, cells were gated on leukocyte using anti-CD45.2

antibody and excluded T cell, B cell, and NK cell using lineages. Macrophages were

isolated using anti-F4/80 antibody. After sorting, RNA isolation was performed using

NucleoSpin RNA XS (Takara Bio, Japan).

Cell culture

Human THP-1 cells were grown in RPMI 1640 (Sigma-Aldrich, St.Louis, MO)

containing 10% FBS and 1% penicillin and streptomycin (Thermo Scientific). THP-1

cells were maintained at 37 ºC in a 5% CO2 atmosphere. THP-1 cells were seeded at

8×105 cells/well in 6-well-plates, and differentiated by addition of 50 nM PMA for 48 hr.

Cells were then washed in PBS and incubated for 24 hr. hAGP (0.5 mg/mL to 2.5 mg/mL)

which was quantified by BCA protein assay was added and cells incubated for a further

48 hr. In the experiments with stimulation by LPS, hAGP (1.0mg/mL) or IL-4 (10 ng/mL)

and IL-13 (10 ng/mL) was added to the cells for 24 hr, and then cells stimulated with LPS

(100 ng/mL) for 2 hr.

12

Western blotting

AGP in plasma and urine was confirmed by Western blotting for each mouse. Both plasma

and urine samples (0.5 µL per lane) were boiled for 3 min at 100 ºC, and separated by

10% SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes

(Immobilon-P; Millipore, Billerica, MA) by wet electroblotting. The membranes were

incubated in ORM1 antibody (1:5000, Proteintech, IL). Antibody signals were detected

with LAS-4000min (GE healthcare).

Quantitative RT-PCR

Total RNA from kidney and liver isolated by the RNAiso plus (TaKaRa Bio, Otsu, Japan)

was reverse-transcribed with the PrimeScriptTM RT Master Mix (TaKaRa Bio).

Quantitative RT-PCR analysis was performed in an iCycler thermal cycler (Bio-Rad

Laboratories, Hercules, CA) using SYBR Premix Ex TaqII (TaKaRa Bio). The

quantitative RT-PCR method has been previously described30 and the primers used are

listed in the Supplemental Table. GAPDH was used as an internal control.

Statistical analyses

The means for two group data were compared by the unpaired t-test. The means for more

than two groups were compared by one-way ANOVA followed by Tukey’s multiple

comparison. A probability value of P

13

Results

Exogenous hAGP administration suppresses proteinuria and renal injury in ADR mice

After adriamycin injection (ADR mice), hAGP (5 mg/mouse) was intraperitoneally

administrated for 5 consecutive days (Figure 1A, treatment schedule). The results show that

urinary albumin to creatinine ratio in ADR mice was significantly reduced from 7 to 21 days in

the hAGP-treated group (Figure 1B). Furthermore, the increased plasma cholesterol level as

observed in ADR group was significantly suppressed by hAGP administration. (Figure 1C).

The renal function of each mouse was measured (BUN and serum creatinine), but no significant

differences were observed among Control, ADR and ADR treated with hAGP group

(Supplemental Figure 1B). Renal histological analyses at day 21 showed that hAGP

significantly suppressed glomerulosclerosis and tubular atrophy in ADR mice (Figure 1D).

AGP distribution in the kidney was confirmed by immunofluorescence staining using anti-AGP

antibody. Fluorescence was largely detected in the glomerulus in control mice, but was

markedly decreased in the ADR treated group. In contrast, similar levels of fluorescence in

glomerulus were observed in control mice and ADR mice administered with hAGP. (Figure 1E).

Since the plasma AGP level is up-regulated during inflammation, we examined the change in

levels of endogenous mouse AGP (mAGP) in plasma of ADR mice by Western blotting.

Although ADR injection increased the expression of AGP mRNA (Orm1, Orm2 and Orm3) in

liver (Figure 1F), plasma mAGP tended to decrease between 7 to 21 days. In contrast to plasma

levels, urinary mAGP was increased in ADR mice (Figure 1G). These data suggest that even

though mAGP production is increased in renal inflammation induced by ADR injection, the

plasma mAGP level did not increase due to its higher urinary excretion.

Exogenous hAGP administration exerts anti-inflammatory effects in kidneys of ADR mice

14

To evaluate the inflammatory condition, kidney sections were stained with anti-F4/80 antibody,

a representative macrophage marker. The results show that hAGP administration significantly

decreases macrophage infiltration (Figure 2A). It has been reported that the expression of TGF-

β increases in damaged glomerular cells including podocytes, while IL-1β is produced by

macrophages infiltrating into the glomerulus.31, 32 Therefore, levels of mRNA for TGF-β and

IL-1β were measured. As shown in Figure 2B and 2C, TGF-β and IL-1β were significantly

decreased in ADR mice treated with hAGP. Interestingly, hAGP administration significantly

increased CD163 mRNA levels and not increased CD206 mRNA levels in kidney. Both of these

molecules are representative M2 macrophage markers (Figure 2D and 2E), while the expression

of iNOS, which is used as a M1 macrophage marker significantly decrease in the hAGP-

administered group (Figure 2F). Moreover, in the hAGP-administered group, there was

significant increased expression of IL-10 compared with control mice (Figure 2G).

Exogenous hAGP administration induces a CD163-expressing macrophage phenotype at

day 7 in ADR mice

Although hAGP administration significantly decreased urinary albumin to creatinine ratio at

day 7 (Figure 1B), neither renal histological damage nor infiltration of macrophages were found

in any of the three groups (control, ADR and ADR+hAGP group) (Figure 3A and 3C).

Compared to control mice, nephrin expressions as a podocyte injury were decreased in ADR

mice treated with/or without hAGP at day 7 (Figure 3B). On the other hands, the localization

of AGP to the glomerulus was increased by hAGP administration in ADR mice at day 7 (Figure

3B). In the same experimental conditions, hAGP administration suppressed adriamycin-

induced TGF-β mRNA expression (Figure 3E), while no significant change in IL-1β mRNA

expression was found in any of the three groups (Figure 3F). Since the infiltration of

15

macrophages were not changed between ADR and hAGP-treated ADR group (Figure 3C), we

isolated macrophages from kidney to evaluate the macrophage phenotype. As shown in Figure

4A, the mRNA expression of CD163 was decreased in macrophages from ADR mice, while

hAGP administration tended to increase the mRNA expression of CD163 (Figure 4A). Next,

we administered hAGP to control mice and measured mRNA expression of CD163 and CD206

in the isolated macrophages. Although the glomerular fluorescence intensities of AGP were not

significantly changed between control and hAGP-treated control mice, hAGP administration

significantly increased a CD163-expressing renal macrophage even in the control mice (Figure

4B).

hAGP treatment switches PMA-differentiated THP-1 cells into CD163-expressing

macrophages

To further study the effect of hAGP on plasticity of macrophage phenotype, phorbol 12-

myristate 13-acetate (PMA)-differentiated THP-1 cells, a macrophage-like cell, were treated

with hAGP. Since hAGP circulates at 0.5 mg/mL in the plasma of healthy humans and levels

increase to 1.0 to 2.5 mg/mL during inflammation, in vitro experiments were performed using

these plasma concentrations of hAGP. Expression of CD163 mRNA was increased by hAGP in

a dose-dependent manner consistent with our previous results.23 In addition, we newly found

that hAGP dose-dependently decreased the expression of CD206 and iNOS mRNA (Figure 5B-

C). These in vitro data are consistent with the changes in renal expression of CD163 and iNOS

mRNA as shown in Figure 2 and 4. Interestingly, hAGP also dose-dependently increased the

expression of IL-10 mRNA (Figure 5D). The changes of CD163 and CD206 expression in

macrophages treated with hAGP were compared to those in IL-4/IL-13 or IL-4-treated

macrophages, a representative M2 macrophage phenotype. IL-4/IL-13 or IL-4 treatment did not

16

result in an increase in CD163 expression in macrophages, while CD206 expression was

significantly increased (Figure 5E and 5F).

The AGP-induced CD163-expressing macrophage phenotype has anti-inflammatory

properties

Next, we compared the phenotype and anti-inflammatory properties of macrophages treated

with AGP or IL-4/IL-13 in inflammation conditions. As shown in Figure 6, hAGP- or IL-4/IL-

13-induced macrophages maintained their phenotypes even after LPS challenge for 2 hr. The

expression of IL-10 mRNA was measured after stimulating with LPS for 2 hr after treatment

with either AGP or IL-4/IL-13. Interestingly, AGP-treated macrophages had a higher ability to

induce IL-10 expression compared with IL-4/IL-13-treated macrophages (Figure 6D).

17

Discussion

In the present study, we found that AGP potentiates the change of macrophage phenotype into

CD163-expressing macrophages with anti-inflammatory function in vivo, thereby reducing

proteinuria, inflammation and renal damage.

Although AGP-induced CD163-expressing macrophages have M2-like properties, they differ

from IL-4/IL-13-induced M2 macrophages (Figure 5). Both CD163 and CD206 are normally

used as markers of M2 macrophages,33, 34 but the changes in expression of each marker are not

always consistent during macrophage activation switch. For example, Porcheray et al. showed

that IL-4 induced CD206 but not CD163 in human monocyte derived macrophages. In contrast,

IL-10 induced CD163 but not CD206 in the same conditions.33 These results are similar to the

characteristics of hAGP-treated macrophages as shown in Figure 5. Moreover, Furuhashi et al.

previously reported that adipose-derived stromal cells induced both CD163+ macrophages and

CD206+ macrophages in crescentic glomerulonephritis rats and CD163+ macrophages had

higher IL-10 expression compared with CD206+ macrophages.26 This observation is agreement

with the present results in which hAGP-induced CD163+ macrophages have higher IL-10

production ability compared with IL-4/IL-13-induced CD206+ macrophages (Figure 6).

Previously, we reported that hAGP increased CD163 and IL-10 via weak stimulation of the

TLR4/CD14 pathway.23 Subsequently, Nakamura et al. reported that ORM1, one of the AGP

variants, induced M2b monocytes in vitro35 and produced IL-10 which is known to be induced

by TLR4 signals. Therefore, the AGP-induced CD163-expressing macrophages found in this

study may correspond to M2b macrophages which are also known as regulatory macrophages.

It is recognized that regulatory macrophages produce high levels of IL-10 which limits

inflammation by suppressing the production and activation of various pro-inflammatory

cytokines. Although IL-4/IL-13 induced macrophages were also effective against inflammatory

18

disease, they have the potential to increase the risk of tissue fibrosis.36, 37 On the other hands,

regulatory macrophages have a low fibrotic potential compared to IL-4/IL-13 induced

macrophages.38 Therefore, it is unlikely that hAGP-induced CD163-expressing macrophages

have the potential to induce renal fibrosis. This is supported by our recent study in which

exogenously administered hAGP suppressed unilateral ureter obstruction-induced renal fibrosis

in a mouse model.27

Administration of hAGP inhibited the urinary albumin in ADR mice between day 7 and 21.

However, renal histological damage and infiltration of macrophages in ADR mice were

markedly weaker at day 7 than at day 21. Reflecting this data, the expression of IL-1β mRNA

was not changed in ADR mice because IL-1β is produced by infiltrating glomerular

macrophages. These results imply that other mechanisms may be involved in the decrease in

urinary albumin in hAGP-treated mice at day 7. The glomerular barrier has both size and

negative charge selectivity. Negative charge selectivity is a result of the endothelial cell surface

layer (ESL) which functions to suppress albumin leakage into urine.39-41 Interestingly, AGP is

localized at the ESL and contributes to the negative charge barrier in glomerulus because AGP

is a highly negative charged protein resulting from the presence of sialylated N-glycans (pI of

2.8-3.8).13 Hjalmarsson et al. reported that the administration of hAGP increased negative

charges at the ESL and improved the podocyte morphology in rats with puromycin

aminonucleoside-induced nephrosis.42 Our study found that the localization of AGP in the

glomerulus was increased by hAGP treatment at day 7 (Figure 3B). Since it was reported that

damaged glomerular cells such as podocytes produced TGF-β, the increased glomerular

localization of AGP might contribute to the inhibition of TGF-β mRNA expression in ADR

mice (Figure 3D). In addition, these data suggest that administered hAGP might be localized

for at least 3 days in the glomerulus and hence might contribute to the suppression of proteinuria

19

at day 7. On the other hands, increased glomerular localization of hAGP was not found in

hAGP-treated control mice (Figure 4B). This result indicates that the accumulation of AGP may

require a decreasing negative charge in glomerulus. Further investigation would be needed to

investigate the role of exogenously administered hAGP on glomerular negative charge

selectivity in the future. In addition, the anti-AGP (ORM1) antibody used in this study has cross

reactivity with human (hAGP) and mouse (mAGP). Therefore, we could not clearly distinguish

whether the localization of AGP in the glomerular coming from endogenous or exogenous AGP

in this experimental condition. Further study may be needed using hAGP or mAGP specific

antibody if these antibodies are commercially available.

In conclusion, the present study indicates that AGP has a unique mechanism in regulating

inflammation and has potential to suppress urinary protein levels by targeting the ESL. The

results showed that endogenous AGP could work to protect against glomerular disease. Since

proteinuria is an independent risk factor for CKD progression and CVD onset, 2, 3 it is

speculated that alleviation of proteinuria and protection against renal tissue damage by AGP

could be a new therapeutic strategy for CKD such as nephrosis and diabetic nephropathy and

CVD. Future investigation would be needed to evaluate if AGP is also protective if given in a

later phase of the disease.

Disclosures

M Tanaka reports personal fees from Torii and personal fees from Kyowa Kirin outside the

submitted work. M Fukagawa reports grants and personal fees from Kyoto Kirin, personal fees

from Bayer Japan, and grants and personal fees from Ono Pharmaceutical outside the submitted

work. The authors declare no conflicts of interest associated with this manuscript.

20

Acknowledgements

This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research

(16K15320), JSPS Fellows (18J23070) from the Japan Society for the Promotion of Science

(JSPS) and KUMAYAKU Alumni Research Foundation.

Author Contributions

Rui Fujimura: Conceptualization; Formal analysis; Investigation; Visualization; Writing -

original draft

Hiroshi Watanabe: Conceptualization; Funding acquisition; Project administration;

Supervision

Kento Nishida: Investigation

Yukio Fujiwara: Investigation; Methodology; Resources; Writing - review and editing

Tomoaki Koga: Data curation; Investigation; Resources

Jing Bi: Investigation; Resources

Tadashi Imafuku: Investigation; Resources

Kazuki Kobayashi: Conceptualization; Investigation

Hisakazu Komori: Conceptualization; Investigation

Masako Miyahisa: Investigation; Methodology

Hitoshi Maeda: Methodology; Writing - review and editing

Motoko Tanaka: Writing - review and editing

Kazutaka Matsushita: Writing - review and editing

Takashi Wada: Writing - review and editing

Masafumi Fukagawa: Funding acquisition; Methodology; Project administration;

Supervision; Writing - review and editing

Toru Maruyama: Data curation; Funding acquisition; Methodology; Project administration;

Supervision

21

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25

FIGURE LEGENDS

Figure 1. Exogenous hAGP administration suppresses proteinuria and renal injury in

ADR mice

(A) Schedule of hAGP treatment. BALB/c mice were administered with hAGP (5 mg/mouse)

for 5 consecutive days after ADR injection. Mice were sacrificed at day 21. (B) Urinary albumin

to creatinine ratio at day 7 and day 21 after ADR injection. (C) Total plasma cholesterol at day

21 after ADR injection. (D) Representative PAS-stained sections at day 21 after ADR injection

and quantitative of evaluation of glomerulosclerosis and tubule damage. Scale bars: 100μm

(Control n=5, ADR, ADR+hAGP n=6) (E) Representative fluorescence photomicrographs

showing AGP localization in kidney at day 21. The glomeruli are enclosed in a white dotted

line. Scale bars: 100μm. Red: AGP (n=4 per group) (F) Expression of mAGP mRNA in liver

was determined by quantitative RT-PCR. (Control n=5, ADR, ADR+hAGP n=6) (G) Level of

endogenous mAGP in plasma and urine determined by Western blot analysis (Control n=3,

ADR n=4). nd: not detected. All data are mean ± SE. *P< 0.05, **P

26

Figure 3. Exogenous hAGP administration induces an M2-like macrophage phenotype at

day 7 in ADR mice

(A) Representative PAS-stained sections at day 7 after ADR injection and quantitative of

evaluation of glomerulosclerosis and tubule damage (Control n=5, ADR, ADR+hAGP n=6).

Scale bars: 100µm. (B) Representative fluorescence photomicrographs showing nephrin and

AGP localization in glomerulus at day 7. The glomeruli are enclosed in a white dotted line.

Green: Nephrin, Red: AGP. (Control n=3, ADR, ADR+hAGP n=5) (C) Representative

micrographs of kidney at day 7 after ADR injection, stained with antibody against F4/80+

macrophages and quantitative of evaluation of F4/80+ cells (n=4 per group). Scale bars: 100µm.

(D, E) Levels of expression of TGF-β, IL-1β mRNAs in kidney at day 7 after ADR injection

were determined by quantitative RT-PCR (Control n=5, ADR, ADR+hAGP n=6). All data are

mean ± SE. *P< 0.05 ; by one-way ANOVA and post hoc Tukey’s multiple comparison.PAS,

periodic acid-Schiff.

Figure 4. Exogenous administration of hAGP induces CD163-expressing renal

macrophages.

(A) The mRNA expression of CD163 and CD206 in renal macrophages at day 7 after ADR

injection (Control n=4, ADR, ADR+hAGP n=6). (B) Representative fluorescence

photomicrographs showing AGP localization in Control and Control+hAGP mice. The

expression of CD163 and CD206 mRNAs in renal macrophages in Control and Control+hAGP

mice (Control n=5, Control+hAGP n=5). All data are mean ± SE. *P< 0.05; by one-way

ANOVA and post hoc Tukey’s multiple comparison or unpaired t-test.

27

Figure 5. AGP treatment switches PMA-differentiated THP-1 cells into M2-like

macrophages

(A-D) The expression of CD163, CD206, iNOS and IL-10 mRNAs in PMA-differentiated THP-

1 cells treated with AGP (0-2.5 mg/mL) for 48 hr (n=6 per group). (E,F) Expression of CD163

and CD206 mRNAs in PMA-differentiated THP-1 cells treated with AGP (1.0 mg/mL) or IL-4

(20 ng/mL) and IL-13 (20 ng/mL) or IL-4 (30 ng/mL) for 24 hr (n=3 per group). All data are

mean ± SE. *P< 0.05, **P

Figure 1 Fujimura R. et al.

ADR

(Day)0 1 2 3 4

hAGP

7 21

Sacrifice

A

Control ADR ADR+hAGPD

****** **

Plasmaurine

Day7 Day21 Day7 Day21

Control ADR

G

E Control ADR ADR + hAGP

F

Glo

mer

ulos

cler

osis

(%)

Dam

aged

Tub

les

(%)

0

10

30

40

0

20

40

60

0

1000

2000

3000

4000

Day7 Day21

** *

** **

Urin

ary

albu

min

/ cre

atin

ine

(mg

/ mm

ol)

B C** *

** **

* P=0.0507

0

100

200

300

400

Tota

l pla

sma

chol

este

rol

(mg

/ dL)

20

**

0

50

100

150

Fluo

resc

ence

Inte

nsity

(% o

f Con

trol

)

5

10

20

25

15

0OR

M1

mR

NA

(Fol

d ch

ange

)

50

100

200

250

150

0OR

M2

mR

NA

(Fol

d ch

ange

)

* P=0.0503

50

100

150

0OR

M3

mR

NA

(Fol

d ch

ange

)

P=0.0504 50

150

0Plas

ma

AGP

(% o

f Con

trol

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200

100

Day7 Day21

5

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0

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e AG

P(In

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ated

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ncity

(X10

3 ) 15

Day7 Day21

nd nd

α1-acidα1-acid

Figure 2 Fujimura R. et al.

Control ADR ADR+hAGPA

* *

B C D

E F

5

4

3

0

20

15

10

5

0

F4/8

0+M

acro

phag

es(×

102

cells

per

hpf

)

IL-1

0 m

RN

A (F

old

chan

ge) G

CD20

6m

RN

A (F

old

chan

ge)

**

CD

163

mR

NA

(Fol

d ch

ange

)

iNO

Sm

RN

A (F

old

chan

ge)

** ***

TGF-β

mR

NA

(Fol

d ch

ange

)

** ** 40

30

10

20

0

200

150

50

100

0

2

1

5

15

10

0

IL-1β

mR

NA

(Fol

d ch

ange

)4

3

1

2

0

** *

5

15

10

0

Figure 3 Fujimura R. et al.

Control ADR ADR+hAGPA

Control ADR ADR+hAGPC

D E

Glo

mer

ulos

cler

osis

(%) 15

10

5

0

Dam

aged

Tub

les

(%)

1.0

0

1.5

2.0

F4/8

0+M

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phag

es(×

102

cells

per

hpf

)

Control ADR ADR+hAGP

TGF-β

mR

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(Fol

d ch

ange

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IL-1β

mR

NA

(Fol

d ch

ange

)

*AG

PN

ephr

in

*

5

4

3

0

2

1

0

50

100

150

Fluo

resc

ence

Inte

nsity

(% o

f Con

trol

)

200Nephrin

*

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resc

ence

Inte

nsity

(% o

f Con

trol

)

0

50

100

150*

*AGP

B

0.5

2

3

4

1

0

0

5

10

15

ControlControl+ hAGP

Figure 4 Fujimura R. et al.

1.0

0

1.5

CD

163

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(Fol

d ch

ange

)

0.5

CD

206

mR

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(Fol

d ch

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)

1.0

0

1.5

0.5

2.0*

3

0

4

CD

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(Fol

d ch

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2

1

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*

A

B

2.0

0.5

2.5

CD

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mR

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(Fol

d ch

ange

)

1.5

1.0

0Control Control

+hAGP

AGP

0

50

100

150

Fluo

resc

ence

Inte

nsity

(% o

f Con

trol

)

Control Control+hAGP

Figure 5 Fujimura R. et al.

0.5 1.0 2.5 (mg/mL)

Normal Inflammation

Plasma AGP concentration (Human)

A

0hAGP 0.5 1.0 2.5 0hAGP 0.5 1.0 2.5

B C

0hAGP 0.5 1.0 2.5

D

0hAGP 0.5 1.0 2.5

*

0

0.5

1.0

1.5

0

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1.5

0

10

20

30

****

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20

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10

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(Fol

d ch

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)

**

0

1.0

1.5

2.0

2.5

CD

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(Fol

d ch

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)

CD16

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old

chan

ge)

CD20

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chan

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iNO

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chan

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IL-1

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chan

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ー

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****

**

****

* ****

** ****

**** **

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10

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IL- 4 IL-13

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*

IL- 4 IL-13

hAGP

LPS

A ****

**

LPS

****

**

Figure 6

B C**

****

D**

****

Fujimura R. et al.

**

hAGP orIL-4 +IL-13

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26 (hr)

**

IL-1

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chan

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0

1

2

3

4

5

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1.0

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2.0

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1.5

0

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20

30

40

CD

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0.5

CD

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IL- 4 IL-13

hAGP

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IL- 4 IL-13

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RN

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IL- 4 IL-13

hAGP

**

000078 Fujimura*equal contribution: RF and HWP#PCorresponding authors:SResults

000078 Fujimura figuresSlide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6